Large-scale electroporation plates, systems and methods of use

ABSTRACT

Described are electroporation plates ( 2 ) that comprise a plurality of energizable electroporation wells ( 4 ) or chambers arrayed in a solid substrate, wherein at least two of the wells ( 4 ) in the plate ( 2 ) can be independently energized. Each well ( 4 ) contains at least two electroporation electrodes ( 12, 14 ) disposed therein, and serves as the vessel in which an individual electroporation reaction can be conducted. Also, described are electroporation systems that use electroporation plates ( 2 ) according to the invention, as well as methods of using such electroporation plates ( 2 ) and systems, for example, to optimize electroporation conditions.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. provisionalpatent application Ser. No. 60/430,738, filed 3 Dec. 2002, entitled“Multiple Well Electroporation Plates”, under 35 U.S.C. §119(e).

TECHNICAL FIELD

This invention relates to electroporation. More particularly, theinvention concerns large-scale (e.g., large volume, multiple well, etc.)electroporation plates, systems for use in conjunction with such plates,and methods relating to the use of such plates.

BACKGROUND OF THE INVENTION

1. Introduction

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication specifically or implicitlyreferenced is prior art.

2. Background

Electroporation is a well-established technique for moving exogenousmolecules, including nucleic acids, drugs, and other compounds, acrossmembranes, including cell membranes and the membranes that formliposomes and other lipid-encapsulated vesicles. It involves theapplication of electric fields of suitable strength across a samplecontaining, for instance, cells to be made competent for theintroduction of molecules of interest. While the mechanism by whichelectroporation functions is not fully understood, in the context ofliving tissue electroporation is known to involve the breakdown of thecell membrane's lipid bilayer, leading to the formation of transient orpermanent pores in the membrane that permit exogenous molecules to enterthe cell by diffusion.

In the context of ex vivo methods for introducing exogenous moleculesinto cells and other lipid-enclosed vesicles, electroporation offersnumerous advantages: it can be used to treat whole populations of cells(or vesicles) simultaneously; it can be used to introduce essentiallyany macromolecule into a cell (or vesicle); it can be used with a widevariety of primary or established cell lines and is particularlyeffective with certain cell lines; and it can be used on bothprokaryotic and eukaryotic cells without major modifications oradaptations to cell type and origin. Additionally, electroporation canbe used on cells in suspension or in culture, as well as cells intissues and organs. Cells can also be electroporated prior to exposureto the molecular species to be introduced into the cell. Indeed, cellscan be rendered highly competent for transfection or transformation byelectroporation, after which they can be stored prior to exposure to themolecules (e.g., expression vectors encoding one or more genes ofinterest) to be introduced.

Typically, ex vivo electroporation is conducted by positioning asingle-channel apparatus that includes at least a pair of electrodes,i.e., a cathode and an anode, in a sample-containing chamber such as adisposable cuvette. See, e.g., Neumann et al., Biophysical Journal, 71,pp. 868-77 (1996). An electric potential is typically applied using agenerator that emits pulses of a high-voltage electric field to asolution or suspension containing a cell population obtained from apatient, and whether the pore formation is reversible or irreversibledepends on such energizing parameters as pulse amplitude, duration, waveform shape, and repetition rate, in addition to the type and developmentstage of the cells. It is believed that pore formation in, orpermeabilization of, the membrane occurs at the cell poles, the sites onthe cell membranes that directly face the electrodes and thus experiencethe highest transmembrane electric field potential. Unfortunately, in agiven electroporation experiment, the degree of permeabilization mayvary with the cell type, and even among cells in a given population.Variation is also frequently seen from experiment to experiment usingthe same sample electrolyte conditions and cell type. Furthermore, sincethe procedure may be performed in large populations of cells whoseindividual properties vary, electroporation conditions typically canonly be selected to address the “average” qualities of the particularcell population.

As mentioned above, electroporation has traditionally been conducted indisposable single chamber cuvettes, which typically have a maximalvolume of about one milliliter (mL) for purposes of electroporation.Such techniques, however, are tedious, labor intensive, and requireoptimization. To date, efforts to increase the throughput ofelectroporation processes have revolved around multi-channel electrodesystems, which have been used for quasi-high throughput introduction ofexogenous molecules into cells in an attempt to limit the need fortransferring cells from culture containers to electroporation cuvettes.A conventional multi-channel electroporation apparatus includes aplurality of pairs of electrodes that can be inserted in respective onesof a plurality of chambers that hold the exogenous materials and thecells. Currently available multi-channel electroporation devices contain8 or 96 pairs of coaxial electrodes (Genetronics, Inc., San Diego,Calif.). These devices are used for electroporation in standard 96-wellplates, which consist of 8 rows and 12 columns of wells and have astandard size of about 8.5 (W) centimeters (cm) by about 12.7 cm (L),with a standard center-to-center spacing of approximately 9.0millimeters (mm) between wells. See U.S. Pat. No. 6,352,853.

While conventional quasi-high throughput electroporation devices forelectroporation of multiple cell samples or populations have foundlimited application, they possess inherent flaws that limit theirwidespread adoption. These flaws include that such devices employ twoseparate components, namely a multi-well plate and an electrode arraythat is inserted into the wells after the various reagents (e.g., asuspension of containing host cells in a suitable buffer and theexogenous molecules to be electroporated into the cells) have beenadded. In such currently available systems, while the multi-well platesmay be disposable, the electrode array is not, and must be cleaned aftereach use, thereby greatly limiting the true throughput and automation ofsuch systems. Another major flaw in the design of such systems is thatthe electrodes of such arrays, after introduction into the wells, cannotbe optimally positioned in the wells, as some clearance space must beleft inserting the electrodes into the wells. As such clearance spacereduces the percentage of the sample exposed to the electric field,electroporation efficiency is necessarily reduced. Also, most electrodearrays for such systems employ multiple cylindrical outer electrodeseach positioned about a central pin electrode. Such a configurationinherently results in electric fields of varying strengths at differentlocations in each well. Another disadvantage of such systems is thatonly a single set of energizing parameters can be investigated on anyone plate, as the electrode pairs in the arrays typically used in suchsystems are not capable of being energized independently from one ormore of the other electrode pairs in the array. Thus, optimizationexperiments require the use of at least one multi-welled plate for eachset of energizing parameters selected. Other flaws are also known tothose familiar with such devices, including the loss of sample due towicking of the sample onto the electrodes.

In addition to the above shortcomings, when large volumes are to beelectroporated, conventional techniques typically rely on “flow through”systems, where a portion of the total volume to be electroporated ismoved into an electroporation chamber and the electroporating pulses areapplied. The chamber is then emptied and refilled as many times asnecessary to electroporate all of the cells in the total volume of cellsto be electroporated. The reasons for such repetition of pulsing areseveral fold. For example, conventional, high voltage electroporationpulse generators can typically deliver energy to only about four 1 mLcuvettes at a given time. An additional drawback is that the requirementfor multiple pulsing on the sample is deleterious to the cells in thesample often resulting in unacceptable death rates of the cells incertain applications where the incidence of molecular transfer into thecells of the sample is known to be low. See, e.g., U.S. Pat. Nos.6,207,488, 5,676,646, and 5,545,130 for descriptions of flow-throughelectroporation devices and their use.

Given the limitations of conventional electroporation devices as theyrelate to throughput and volume, the need clearly exists for devices andsystems suitable for high throughput and other large-scale applications.The instant invention addresses these and other needs in theelectroporation arts, as described below.

Definitions

Before describing the instant invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification, asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings.

As used herein, the term “contacting” refers to any method of exposing ahost cell to an exogenous molecule. For example, a host cell, orpopulation of host cells, can be immersed or bathed in anelectroporation buffer solution containing one or more species ofexogenous molecules. Contact between a host cell and an exogenousmolecule may occur either before or after application of electricimpulse(s) via electroporation. In response to such contact, anexogenous molecule is “introduced” into a host cell when the exogenousmolecule enters the host cell and exhibits either a transient or stableeffect, for example, expression of a heterologous nucleic acid moleculein the host cell (e.g., the production of biologically green fluorescentprotein encoded by an expression vector).

“Energizing parameters” refer to the particular energizingcharacteristics delivered by a pair of electroporation electrodes in agiven experiment. Such parameters include pulse amplitude, duration,wave form shape, repetition rate, and interval between pulses.

An “exogenous molecule” refers to any molecule intended for introductioninto to a host cell. Exogenous molecules include nucleic acid molecules(e.g., expression vectors carrying one or more genes or heterologousnucleic acids encoding proteins or polypeptides to be expressed,anti-sense molecules, ribozymes, small interfering RNAs, etc.), smallmolecule drugs, and other chemicals that somehow alter, transiently orpermanently, the cell or its function(s), including the biochemicalactivity of any protein or enzyme in the cell.

The term “genetically modified” refers to the introduction of one ormore heterologous nucleic acids into one or more host cells. A“heterologous nucleic acid” refers to a nucleic acid molecule thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its original form or will result in anincrease in the copy number of the introduced nucleic acid.

A “host cell” is any cell into which an exogenous molecule can beintroduced. Host cells include eukaryotic and prokaryotic cells.Eukaryotic host cells include animal, plant, and fungal cells. Preferredanimal cells include those from mammals (e.g., bovine, canine, equine,feline, murine, ovine, and porcine animals), including humans andprimates, fish (e.g., zebrafish, salmon, trout, and other commerciallyimportant species), insects (e.g., the fruit fly, mosquitoes, bees),arachnids, birds, crustaceans, and mollusks, as well as cell linesderived from cells of any of the foregoing organisms. Preferred plantcells include those from crop plants such as cereal grains, as well asornamental plants and trees grown for lumber production. Preferredfungal cells are yeast cells. Preferred prokaryotic cells are bacterialcells, particularly species useful in molecular biology. Host cells intowhich a heterologous nucleic acid is introduced are referred to as“recombinant host cells”. Host cells also include artificial cells,liposomes, and any other lipid-encapsulated vesicle into which anexogenous molecule can be introduced by electroporation. For purposes ofbrevity, “host cells” may also be referred to herein simply as “cells”.

Performing a method “in vitro” refers to performing the method outsideof an organism, and includes the concept of ex vivo methods, wherein,for example, a sample of cells is removed from a patient andelectroporated using the devices and methods of the invention tointroduce one or more species of exogenous molecules into the cells,after which the treated cells are reintroduced into the patient.

“Large-scale” means either or both that the electroporation plates ofthe invention are amenable to high throughput and/or large volumeapplications. Here, “high throughput” refers to the ability to performtwo or more, preferably, 4, 16, 32, 64, 96, 192, 288, 384, 576, 768,672, 1536, 3072, or 6144 or more identical or different electroporationexperiments on the same electroporation plate.

A “large volume” application refers to a volume that exceeds the volumeof a conventional electroporation cuvette or other vessel for conductinga single electroporation experiment. Typically, conventionalelectroporation cuvettes or other vessels provide of volume of up toabout 1 mL, although vessels that may contain up to about 10 mL areknown, particularly in the context of flow-through electroporationdevices. In the context of this invention, the vessel may contain avolume from as small as about 1 microliter (uL) to about 10 mL or more.Preferred large volume applications allow for the electroporation ofcell-containing solutions having a volume of at least about 5 mL,preferably at least 10 mL to about 100 mL or more.

As used herein, the term “nucleic acid” or “nucleic acid molecule”refers to a polymer of deoxyribonucleotides or ribonucleotides (ineither case, a “polynucleotide”) in the form of a separate fragment oras a component of a larger construct. Nucleic acids may be eithersingle- or double-stranded, and include non-naturally occurring bases orbackbone structures. Such molecules include DNA, RNA, cDNA, antisense,ribozyme, and triplex forming molecules. Nucleic acids can be naturallyoccurring or synthetic, and include oligonucleotides. DNA encodingproteins or polypeptides utilized in the methods of the invention can beassembled, for example, from cDNA fragments or from oligonucleotidesthat provide a synthetic gene that is capable of being expressed in arecombinant transcriptional unit. Polynucleotide or nucleic acidsequences of the invention include DNA, RNA, and cDNA sequences.

A “patentable” composition, process, machine, or article of manufactureaccording to the invention means that the subject matter satisfies allstatutory requirements for patentability at the time the analysis isperformed. For example, with regard to novelty, non-obviousness, or thelike, if later investigation reveals that one or more claims encompassone or more embodiments that would negate novelty, non-obviousness,etc., the claim(s), being limited by definition to “patentable”embodiments, specifically exclude the unpatentable embodiment(s). Also,the claims appended hereto are to be interpreted both to provide thebroadest reasonable scope, as well as to preserve their validity.Furthermore, if one or more of the statutory requirements forpatentability are amended or if the standards change for assessingwhether a particular statutory requirement for patentability issatisfied from the time this application issues as a patent to a timethe validity of one or more of the appended claims is questioned, theclaims are to be interpreted in a way that (1) preserves their validityand (2) provides the broadest reasonable interpretation under thecircumstances.

“Plant” refers either to any whole plant, a plant part, a plant cell, ora group of plant cells, such as plant tissue, for example. The classesof plants whose cells can be used in conjunction with theelectroporation plates of the invention include any higher plant, bethey monocotyledonous or dicotyledonous plants, and any ploidy level,including polyploid, diploid, and haploid. “Monocotyledonous plants”, or“monocots”, include asparagus, field and sweet corn, barley, wheat,rice, sorghum, onion, pearl millet, rye and oats. Examples of“dicotyledonous plants”, or “dicots”, include tomato, tobacco, cotton,rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa,clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli,cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach,cucumber, squash, melons, cantaloupe, sunflowers, and variousornamentals.

“Plant cell” refers to an intact cell of any plant, including a cellfrom a leaf, callus, embryo, or seed, as well as any gamete-producingcell and any cell that regenerates into a whole plant. Thus, a seedcomprising multiple plant cells capable of regeneration into a wholeplant is included in the definition of a plant cell. In this context,term “intact” refers to a single cell or group of single cells whichform a tissue, wherein the cell(s) have undamaged or untreated cellwall(s), as compared to protoplasts.

A “plurality” means more than one.

SUMMARY OF THE INVENTION

It is an object of this invention to provide large-scale electroporationplates, and systems and kits employing such plates, to overcome thedeficiencies of current approaches for introducing exogenous moleculesinto cells, particularly in vitro. Another object of the invention is toprovide methods of using such electroporation plates, for example, tointroduce an exogenous molecule into a host cell in vitro.

Thus, one aspect of the invention concerns electroporation plates thatcomprise a plurality of energizable electroporation wells arrayed in asolid substrate, wherein at least two of the wells in the plate can beindependently energized, i.e., when energy is provided to theelectroporation electrodes in one of the two wells, the electrodes inthe other well need not be energized, although they may be. Each wellcontains at least two electroporation electrodes (at least one anode andat least one cathode) disposed therein, and serves as the vessel inwhich an individual electroporation reaction can be conducted.

In preferred embodiments of this aspect, the wells of an electroporationplate are arrayed in a series of at least two rows and two columns. Inparticularly preferred embodiments, the number of rows differs from thenumber of columns. Typically, but not essentially, the ratio of rows tocolumns is about 2:3. In some preferred embodiments, the plates of theinvention comprise 96 electroporation wells arrayed in 8 rows and 12columns. In other preferred embodiments, the plates comprise 384electroporation wells arrayed in 16 rows and 24 columns. Other preferredplate configurations contain 192, 288, 576, 672, 768, 1536, 3072, or6144 wells. To facilitate automated handling and compatibility withexisting plate-based high throughput systems used in the biologicalsciences, it is preferred that the overall dimensions of the plates(length, width, and height) be the same plate to plate, regardless ofthe number of rows, columns, and electroporation wells, as compared toconventional, non-electrifiable multi-well plates (e.g., as arecurrently used in automated high throughput compound screening systems).

In many embodiments, particularly in plates having fewer than about 384electroporation wells, the wells are substantially cylindrical orrectangular boxes, in either case with one end, at the top of the plate,being open. In other embodiments, especially in those having largenumbers of electroporation wells, the wells tend to be substantiallyrectangular boxes with one open end (at the top), such that when viewedfrom the top, their length and width dimensions are substantiallysimilar (yielding a square shape), with the depth dimension beingdifferent, typically larger, than the length and/or width dimension. Ofcourse, wells having any suitable geometric shape may be employed in thecontext of the invention, although preferred well configurations provideat least two spaced, substantially parallel walls upon which one or moreelectrode pairs are disposed. In any event, it is preferred that theelectroporation wells of a plate have substantially uniform dimensionswhen compared well-to-well. Such consistency in terms of shape serves toprovide a substantially uniform electroporation response from well towell, all else being equal. As electroporation wells are typically openat the top (absent a removable cover plate, seal, or the like), eachwell has only a bottom wall and at least one sidewall (one in the caseof a cylindrically shaped well, four in the case of a rectangle- orsquare-shaped well, etc.). As will be appreciated, to facilitatemanufacture, for example, by injection molding techniques, when viewedfrom the side, a well tends to be slightly tapered, with the upperopening having dimensions (i.e., lengths and widths, or diameters)slightly larger than those at the bottom of the well, therebyfacilitating removal of the plate from a mold.

As will be appreciated, for plates having standardized outer dimensions,increasing the number of electroporation wells typically results in adecrease in individual well volume. In some embodiments of theinvention, an electroporation plate will contain relatively few wells(e.g., 96 or even as few as 2-12), enabling each well to have volume ofabout 1 milliliter (ml), in the case of a 96 well plate, and 10 ml inthe case of 12 wells. Preferred well volumes range from about 10 mL todown to about 1 microliter (uL), although smaller volumes are possible,particularly if, for example, photolithographic techniques from thesemiconductor fabrication industry are adapted to generate platescontaining electroporation wells having volumes on the microliter, andeven nanoliter, scale.

Preferably, in each electroporation well (or chamber) of anelectroporation plate according to the invention, one of theelectroporation electrodes for that well will be positioned, ordisposed, opposite to the other electroporation electrode for that well.Preferably, the electrodes in a given well are substantially parallel toeach other to facilitate generation of a uniform electric field in thewell when its electrodes are energized in the presence of anelectrically conductive solution. In this context, substantiallyparallel means +/−10%, preferably 5%, and even more preferably, 1% orless. Electrodes may be disposed in the wells in any suitable direction,although preferably the electrodes are horizontally or verticallydisposed, as viewed when the plate is placed on a surface to receive asample of cells. Electrodes may, in any given well in, for example,large volume wells, also comprise a plurality of individual electrodes(preferably arrayed in pairs of opposed electrodes, one being an anode,the other being a cathode) that may be energized together or insuccession and in a particular pattern facilitating the substantiallyuniform distribution of electric fields through out the well.

Electroporation electrodes are preferably integrated into thesidewall(s) of a well in a fluidly sealed manner (to prevent leakage ofliquid after it is added to the well) such that a conducting surfacethereof is exposed to a sample placed in the well. Alternatively, toform an electrode, electrode material (e.g., gold) can be deposited,preferably as a thin film, or layer or composite of thin films, on asidewall of the well. Processes for deposition of electrode materialinclude dipping, plating (electroless or electrolytic), spraying, andvapor deposition. Electrodes (or portions thereof to be exposed to hostcells) are preferably made of biocompatible, electrically conductivematerials. Examples of such materials include gold, aluminum, titanium,or a non-metallic, electrically conductive composite such as graphite(or a polymer filled or sufficiently doped with an amount conductivematerial sufficient for the composition to serve as an electrode usefulin the practice of the invention).

In certain embodiments, the electrodes comprise a composite of multipleelectrode materials, e.g., copper, nickel, and gold, each of which isapplied to the solid substrate at different times, thereby building uplayers of electrode material. Preferably, in such composites, theoutermost layer of electrically conductive material (i.e., that to beexposed to cells) is biocompatible. The electrode material(s) in thelayer(s) below the outermost layer may also be biocompatible, althoughfactors such as, for example, cost, compatibility with the underlyingsubstrate or adhesion layer, if any, ease of manufacture or application,and the material's properties as a conductor may influence the skilledartisan's material selection for a given application.

When applied to a sidewall, an electrode preferably covers as much ofthe sidewall as is required to generate a substantially uniform electricfield in the well (or volume between the electrodes) upon beingenergized in conjunction with its complementary electrode(s). As will beappreciated, in large-scale applications where a plurality of electrodesets (e.g., pairs) that are energized at different times are employed,the substantially uniform electric field is generated in the volume ofcell-containing solution between the electrodes that comprise theparticular electrode set being energized, not necessarily in the entirechamber. Preferably the electrodes cover as much of the sidewalls aspossible, as this allows a wide range of solution volumes, up to themaximum for the given well, to be used, in addition to allowing theuniformity of the electric field generated between the electrodes to bemaximized.

Each electrode in an electroporation plate is connected to a conductor(e.g., an electrically conductive wire or contact point) that can carryenergy to the particular electrode from a power source (e.g., a pulsegenerator) that can be connected to the device. In certain preferredembodiments, the electrodes for the electroporation wells in a givencolumn (or row) are interconnected in series or in parallel such thatthey can be energized, or addressed, simultaneously, while theelectrodes for the electroporation wells in other columns (or rows) ofthe plate not electrically connected to the energized wells will not beenergized. In other preferred embodiments, the electrodes for each wellof an electroporation plate can be independently addressed, thusallowing each well of the plate to be energized independently of all ofthe other wells in the plate. Of course, in a plate having independentlyaddressable wells, it may be desirable to similarly energize two or morewells, for example, to generate statistics related to the transformationefficiency of the particular energizing parameters.

The electroporation plates of the invention are made from any suitablesolid substrate. In certain embodiment, the solid substrate is made froma homogenous composition of a single material, while in certain otherembodiments, it can be made from combinations of different materials.Particularly preferred substrates are plastics, since plastics areinexpensive, easily formed into desired shapes (for example, by variousmolding processes, such as injection molding), resistant to breakage,have desirable optical properties, and are easily machined, if desired.In preferred embodiments, all or a portion of the solid substrate willbe transparent to light of one or more wavelengths (e.g., wavelengths inthe visible electromagnetic spectrum). In other embodiments, all or aportion of the solid substrate will be translucent, while in still otherembodiments, all or a portion of the solid substrate will be opaque.

Electroporation plates according to the invention also include at leastone electrical connection that allows energy to be delivered to theelectrodes in the plate. Any suitable electrical connection can be used,including electrical connections that employ pins and sockets. Inparticularly preferred embodiments, a plurality of contact pad-typeelectrical connections is provided, with each electrode (or series ofelectrodes to be energized with the same parameters) being connected toa conductor energized through a separate contact pad. Thus, each contactpad is electrically connected with at least one electrode, therebyallowing the electrode(s) to be energized, if and as desired. The exactnumber of contact pad connectors depends on the number ofelectroporation wells (or complete or partial rows or columns of wells)that are to be independently energized, as well as whether otherelectronic controls (e.g., switches) are provided in a particularcircuit.

The electrical connection(s) on an electroporation plate allow energy tobe supplied to the one or more electrodes present in the plate, asdirected by a controller that directs which, if any, of the electrodesin the plate are to receive energy from the power supply (e.g., a pulsegenerator). The controller can be built into the power supply or,alternatively, it can be a separate device.

Electroporation plates according to the invention may also contain atracking or inventory element, for example, a series of opticallyreadable bar codes that provide a unique identifier, so that aparticular plate can be tracked in an automated or semi-automatedsystem.

A related aspect of the invention concerns electroporation platesconfigured to receive a large volume of a cell suspension to beelectroporated. In embodiments of this aspect, the plate typicallycomprises one to about 50, preferably 1 to fewer than about 12, separatechambers. Each chamber may be open at the top, although this notessential. The chamber(s) may also include one or more ports to allowliquid to flow into and/or out of each chamber. Each chamber may alsocontain one or more internal baffles to limit or control fluid movementwithin the chamber. In some embodiments, electrodes can be positioned onthe baffles. In any event, each chamber comprises at least one, andpreferably a plurality of electrode sets disposed on the walls of thechamber. The electrodes comprising each electrode set are disposed onopposite sides of the chamber. Each electrode set is comprised of aplurality of electrodes, preferably an even number of electrodes (e.g.,2, 4, 6, 8, 10, 12, or more) wherein there is an equal number of anodesand cathodes, although the invention contemplates electrodes sets thatcomprise an odd number of electrodes (e.g., 1, 3, 5, 7, 9, 11, 13, ormore). Indeed an electrode set may comprise as few as one electrode, inwhich event the electrode will be used in conjunction with anotherelectrode set configured accordingly. Particularly preferred areelectrode sets that comprise a pair of electrodes (e.g., one anode andone cathode). Each electrode set may be energized independently of theother electrode sets for a given chamber. Similarly, in embodiments thatcomprise multiple chambers on a single plate, an electrode set in two ormore of the chambers may be energized at the same time or at differenttimes.

During electroporation in a particular chamber that comprises aplurality of electrode sets, it is preferred that the electrode sets beenergized sequentially. In those embodiments where the electroporationcomprises a series of pulses between electrodes in each electrode set,it is preferred that the series of pulses applied between the electrodesof a given electrode set be completed before the electrodes of anotherelectrode set in the chamber are energized, preferably using the sameparameters as were used to energize the preceding set(s) of electrodes.By “sequentially” in this context, it is contemplated that, for example,adjacent opposed pairs of electrodes within an elongate well will be“sequentially” energized from one end of an elongated well to the other.Alternatively, the pulsing may be from the center portion of the well tothe outer ends of the well.

Another related aspect of the invention concerns kits comprising anelectroporation plate according to the invention wherein at least one,some, or preferably, all, of the wells of the plate contain an aliquotof electrically competent host cells. “Electrically competent” refers tohost cells that readily uptake exogenous molecules in the presence, orfollowing the application, of a suitable electric field. Typically, thecells will be suspended in a buffer suitable for storage and transportand in which the cells will remain viable for at least 24 hours,preferably 1-3 days, and even more preferably at least 4-7 days. As willbe appreciated, different cells may require buffers containing differentcomponents. Moreover, it may be desirable to ship cells in a frozenstate, in which event a different buffer may again be required.Accordingly, the selection of buffer and cell combinations is left tothe artisan's discretion. In order to ship cell-containing plates, asuitable plate cover (e.g., a removable foil or plastic cover) ispreferably also provided. To prevent loss of a cell suspension (inliquid or frozen form, as the case may be) from individual wells, it ispreferred that the cover individually seal each well. Cell-containingplates may be packaged individually or in bundles of two or morecell-containing plates. A package insert will typically also be providedin each package.

Another aspect of the invention concerns electroporation systems thatemploy an electroporation plate according to the invention. At aminimum, such systems will comprise at least one such electroporationplate and a power supply adapted for connection to the electricalconnector(s) of the electroporation plate. The power supply will alsoinclude a controller or, if not, the system will include a controller asa separate device. The controller directs energizing of the plate'svarious electrodes, preferably according to a preset program, which maybe user-defined or factory-provided (for example, selected from a menulisting a plurality of pre-programmed sets of energizing parameters).Such a system may optionally include a plate handler configured to holdthe electroporation plate during operation of the electroporationsystem. In preferred embodiments, the plate handler comprises anelectrical connection system compatible with the set of electrodes andconductors in the particular plate such that when a plate is placedtherein, the appropriate electrical connections are made, therebyallowing the desired energizing parameters to be delivered to theelectrodes in the independently addressable wells in the plate, or inthe case of a large-volume plate, to each of the electrode groupings(e.g., opposed pairs of electrodes) that are to be simultaneouslyenergized (e.g. an opposed pairs set). As will be appreciated, one ormore of the power supply, controller, and plate handler may beintegrated into a single device, or into a collection of devices thatnumber fewer than the various functions provided by a stand alone powersupply, controller, or plate handler.

In other embodiments of this aspect, the system further includes one ormore plate readers to collect data from the electroporation wells orchamber(s) of the plate. In high throughput systems that use multi-wellplates, the reader(s) is(are) typically in a different location. Forinstance, after electroporation occurs, for example, in a plate handler,the plate may be moved (e.g., by a robot or other device) to a stationcontaining the reader. Data collection then occurs. In some embodiments,such a system may also include an incubation station, where the plate(and the electroporated cells) is incubated under desired conditionsprior to being moved to a reading (or data collection) station.

To collect data, a plate reader employs a reading device compatible withthe data that will be generated from the electroporation experiment(s)performed in an electroporation plate. For example, if the exogenousmolecule introduced into a host cell is a nucleic acid molecule encodinga reporter gene whose expression product is a protein capable ofgenerating fluorescence, a luminomitor may be used to collect data onhow much luminescence was emitted from a particular well. Similarly,other species of exogenous molecules may be labeled, as desired,typically with a moiety compatible with the detection system employed.If different electroporation conditions, buffers, reactantconcentrations, etc. are used in different wells, different resultscould be expected. Other reading devices include spectrophotometers (forexample, to measure differences in absorbance of a specific wavelength,or range of specific wavelengths, of light in a suspension of host cellsinto which an exogenous molecule was introduced) and machine visiondevices (for example, to assess cell adherence followingelectroporation-mediated introduction of an exogenous molecule into thehost cells in the suspension), although any detection device capable ofdetecting an effect on cells that is mediated by an exogenous moleculemay be employed.

In those embodiments employing a plate reader, it is preferred that theelectroporation system also include a data storage device to store datacollected by the plate reader for later review and analysis. Anysuitable data storage device can be used.

Optionally, an electroporation system will also include a plate storagefacility to store electroporation plates during the course or followingcompletion of an electroporation experiment, preferably after a platereader has collected data.

In other embodiments of this aspect, an electroporation system furthercomprises an optimization computer adapted to optimize electroporationconditions, alone or in conjunction with other experimental conditions,using electroporation data stored in the memory.

Yet another aspect of the invention relates to methods of introducing anexogenous molecule into a host cell, comprising using electroporation tointroduce the exogenous molecule into a host cell present in asuspension contained in an electroporation well of an electroporationplate according to invention. Here, the use of electroporation to“introduce an exogenous molecule into a host cell” refers to renderingthe host cell capable of taking up the exogenous molecule usingelectroporation. As a result of electroporation, the exogenous moleculeis then able to diffuse into the host cell. Of course, techniques suchiontophoresis can also be employed in order to further enhance theuptake of exogenous molecules into cells which have been electroporated.In certain embodiments, the exogenous molecule is a nucleic acidmolecule. In other embodiments, it is a small molecule drug. It will beappreciated that more than one species of exogenous molecule may bepresent in a given electroporation reaction.

In the instant methods, the host cell typically is a eukaryotic cell ora prokaryotic cell. Preferred eukaryotic host cells include animal cellsand plant cells. Particularly preferred animal cells include mammaliancells (e.g., human and primate cells, as well as cells from bovine,canine, equine, feline, murine, ovine, and porcine animals), insectcells, fish cells, bird cells, arachnid cells, mollusk cells, andcrustacean cells. Particularly preferred plant cells include cells frommonocotyledonous or dicotyledonous plants. Also preferred are cell linesdeveloped from any of the foregoing types of cells.

Other features and advantages of the invention will be apparent from thefollowing drawings, detailed description, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and embodiments of the present invention willbecome evident upon reference to the following detailed description andattached drawings that represent certain preferred embodiments of theinvention, which drawings can be summarized as follows:

FIG. 1 is an illustration of a multi-well electroporation plateaccording to the invention. Depicted is a 96-well plate (2). Wells (4)are organized into eight rows (6) and twelve columns (8). The topsurfaces (10) of each row are plated with a conductor to carry current.Extending into the wells are electrodes (12 and 14) connected to theconductors. Conductors for anodes are located on one side of a row,cathodes and their conductors on the other side. In this embodiment,electrical connection to an external energy source occurs at the edge ofthe plates, where electrical contacts with a power supply are made atthe distal regions (16 and 18) of the connectors for anodes andcathodes.

FIG. 2 depicts various components of a representative electroporationsystem for multi-well electroporation plates according to the invention.

FIG. 3 depicts various representative embodiments of large volumeelectroporation plates according to the invention. FIG. 3A is anillustration of a large-scale electroporation plate (30) according tothe invention, wherein the plate is suitable for large volumeapplications and comprises three identical electroporation chambers(32), each of which can hold up to about 15 mL of cell suspension. Ashown, each of the electroporation chambers (32) are mounted on a baseplate (30). Each chamber contains an inlet port (34) and an outlet port(38), as well as a vent (36) to facilitate filling and emptying of thechamber. Also, in the depicted embodiment, each chamber has an ergonomicportion (40) to facilitate handling.

In the illustration shown in FIG. 3B, one of the chambers of theelectroporation plate depicted in FIG. 3A is shown in a cutaway view toshow a representative layout of electrodes. Here, three electrodes (39)disposed on one wall of the inside of chamber the (32) are represented,and each electrode on the wall is separated from another electrode by aninsulating portion of the wall (41). In a chamber, each of theseelectrodes (39) would be paired with an electrode (39, not shown) on theopposite wall, and each electrode pair would be independentlyenergizable. Preferably, the electrodes of each electrode pair are thesame size and are disposed directly opposite one another. The electrodesof an electrode pair are also preferably spaced such that the surface ofone electrode is parallel to the surface of the other electrode toensure generation of electric fields of substantially equal strengthbetween the electrodes, regardless of the position between theelectrodes where field strength is measured. In the embodiment shown inFIG. 3B, the floor (37) of the solution-containing portion of thechamber (32) is tapered in the direction of the outlet port (38) toassist in draining a cell suspension from the chamber.

FIG. 3C shows a cross section of an alternative embodiment of thechambers (32) of FIG. 3A wherein the electrodes (39) are disposedsubstantially horizontally, as opposed to vertically. As in theembodiment of FIG. 3B, the electrodes (39) depicted on one of the innerwalls of the chamber are separated from one another by a smallinsulating portion (41) of the wall.

FIGS. 3D and 3E illustrate embodiments wherein the chamber (32) has aseries of internal baffles (42 in FIG. 3D, 44 in FIG. 3E) thatoptionally may be included in the device. When present, the bafflesserve to limit the movement of liquid in the sample-containing portion(45) of the chamber (39). In the depicted embodiments, each chamber alsohas a sample inlet port (34) and a sample outlet port (38) to allow oneway sample flow into and out of the chamber. FIG. 3D shows across-section taken through a chamber (32) to show a representativeconfiguration of baffles (42). As depicted, each baffle extends downwardfrom the top of the chamber, leaving an opening (43) between the bottomsurface (37) of the chamber (32) and the lower face (48) of the baffle.Preferably, when multiple baffles are included in a chamber in this way,the opening below one baffle is the same size as the openings beneaththe other baffles, although configurations having multiple baffles withdifferently sized openings are contemplated by the invention. As will beappreciated, the baffles may also contain one or more additionalopenings of various shapes and sizes over their length, and they may beattached to one or more sides of the chamber in addition to beingattached to the top of the inside of the chamber.

FIG. 3E shows a related embodiment wherein the chamber (32) containsseveral baffles (44) extending from the side walls of the inside of thechamber in staggered fashion such that one baffle (44) leaves an opening(43) between its face (47) having the closet approach to the oppositewall face (49). As will be appreciated, any suitable embodimentcontaining one or more baffles, regardless of configuration, arecontemplated by the invention. Indeed, the baffles may also containelectrodes (not shown).

FIG. 3F shows another representative embodiment of a chamber (32) for alarge-scale electroporation plate (30) according to the invention thatis suitable for large volume applications. In particular, FIG. 3Fillustrates a top-down view of a single chamber of such a plate thatcomprises four electrode pairs (48 a-d), with the anode (52) of eachelectrode pair being disposed directly opposite the cathode (50) of thepair. In the depicted embodiment, each electrode is integrated into anddisposed vertically in a wall of the chamber directly opposite the othermember of the particular electrode pair. On each of the walls that housethe electrodes, the electrodes are separated from one another by aninsulating portion (41) of the particular wall. The plate and chambersalso comprise conductors (not shown) that connect each of the anodes andcathodes in a particular electrode set to electrical contacts that canbe connected to a power supply (not shown) for energizing theelectrodes. In the depicted embodiment, each electrode set (here, anelectrode pair) may be energized independently of the other electrodesets.

As those in the art will appreciate, the embodiments represented in theattached drawings are representative only and do not depict the actualscope of the invention. For example, the various components of anelectroplate plate according to the invention may be arrangeddifferently or include additional and/or different components.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail, it is understoodthat the invention is not limited to the particular electroporationplates, systems, and methodologies described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the invention defined by the appended claims.

1. Introduction.

At its core, the present invention relates to large-scaleelectroporation plates. In certain preferred embodiments, these plateshave at least two wells, and as many as 96, 192, 288, 384, 576, 672,768, 1536, 3072, 6144, or more identical wells, that containelectroporation electrodes that can be independently energized oraddressed. In other preferred embodiments, an electroporation plateaccording to the invention comprises at least one, and as many as 2-30or more, large volume chambers for electroporating cell-containingsuspensions. In such embodiments, each chamber comprises a plurality ofelectrode sets that can be independently energized or addressed.

It is understood that, in the context of this invention, “independentlyenergized”, “independently addressed”, or the like refers to thecapability of a pair of electroporation electrodes (or larger number ofelectrodes comprising an electrode set, e.g. opposed pairs ofelectrodes) to receive energy separate and apart from any other pair ofelectroporation electrodes in the plate. This capability is a functionof the plate design, and can be accomplished many ways. For example,each electroporation electrode pair may be separately wired from theother electrode pairs, where each electrode pair is part of its ownseparate circuit comprised of the electrodes and the conductors thatconnect the electrodes to the electrical contacts for connection to apower supply. Alternatively, a plurality of electrodes, preferablyelectrode pairs, can be configured as elements of the same circuit, witheach electrode pair having a switch associated with it. Thus, a switchwill determine whether a particular electrode pair is energized when thecircuit is energized. Of course, plates having combinations of suchcircuits can also be made. When the electrode pair (or set) for singlewell (or chamber) can be independently addressed, the well (or itselectrodes) is (are) said to be “independently addressable”. Likewise,in other embodiments, where the plate is designed so that several, butnot all, electrode pairs or sets are part of one circuit, and thus canbe energized together (i.e., at the same time, without switchactuation), those electrode pairs (and their corresponding wells) aresaid to be independently addressable, as compared to other electrodes(and wells) in the plate.

In addition to electroporation plates and kits comprised of such platesloaded with electrically competent host cells, the invention alsoconcerns electroporation systems that use such plates. At a minimum,Such an electroporation system will comprise a plate handler thatenergizes the circuit(s) present in an electroporation plate. Thus, aplate handler will comprise a power supply to provide the energy neededto energize a plate's electrodes, as well as a controller that directswhen and how each circuit will be energized (and, if present, whichswitch or switches will be opened or closed). The controller and powersupply may be separate units, although preferably these functionalitiesreside within a single device. An electroporation system can alsoinclude one or more plate readers, data storage devices, and/orcomputers (e.g., to optimize electroporation parameters, manage platemovement and handling, etc.).

Preferred embodiments of these and other aspects of the invention arethoroughly described below.

2. Electroporation Plates.

This invention concerns electroporation plates, articles of manufactureuseful in performing electroporation experiments. With regard toembodiments useful for high-throughput applications, the plates eachcomprise a plurality of electrifiable wells, at least two of which canbe independently energized. The wells are disposed in a solid substrate,and may be introduced at various stages of manufacture. To electrifyeach well, a at least two electrodes (an anode and a cathode) arepermanently disposed therein. As with the wells themselves, theelectrodes may be introduced at various stages of manufacture. Theelectrodes are energized using an external power supply (often referredto in the electroporation field as a “pulse generator”) that can beoperatively coupled to the plate. Similarly, in embodiments where theplates comprise one or more large volume chambers, each chambercomprises a plurality of electrode sets that can be independentlyenergized. Each of the electrodes in an electrode set is preferablydisposed in a wall of the chamber, which is typically made from a solidsubstrate. As will be appreciated, chambers can be made separately andthen be attached to a support member at an appropriate stage.Accordingly, the electrical circuitry for connecting the electrode setsto a power supply can be integrated solely in the body of the chamberitself or, alternatively, it can include components some of which residein the chamber and some of which reside on the support. In otherembodiments, the chambers and support are manufactured as an integratedunit, in which event the completed unit will comprise the conductors andelectrical connections necessary for the electrodes of the device to beenergized by a power supply.

a. Design.

As described herein, a high throughput electroporation plate accordingto the invention comprises a plurality of electrifiable wells, at leasttwo of which can be independently energized. Because such plates arelikely to be used in conjunction with automated, high throughputscreening systems, it is preferred that the plates be designed tooperate in such an environment, although such compatibility is notessential. Developers of hardware for most high throughput biomolecularscreening systems rely on standards published by the Society forBiomolecular Screening (Danbury, Conn.) and approved by the AmericanNational Standards Institute, Inc. Among other things, such standards(be they draft, interim, or formally adopted) have defined a set ofcommon dimensions for plates to be used in these systems. Generally,standards exist for plate footprint dimensions, height dimensions,bottom outside flange dimensions, well positions (for example, for 96,384, and 1536 well plates), and side-wall rigidity. Plates of thesedimensions may be readily handled by robotic systems and automatedhardware platforms in use in many pharmaceutical, biotechnology, andagricultural biosciences corporations, as well as academic and otherresearch institutions throughout the world. To facilitate widespreadadoption of the instant electroporation plates for high throughputapplications, it is desirable that the plates conform to these or laterdeveloped standards to support automation and cross-platformcompatibility.

Preferred examples of such embodiments include those wherein anelectroporation plate comprises a plurality of wells organized incolumns and rows. In some of these embodiments, two or more of the wellsin a given column (or row)(which may or may not be all of the wells inthe column) are energized together, but independently of the wells inother columns (or rows) of the plate. In others, the electrodes of allof the wells of a given column or row will be energized together, butindependently from, the other electrodes of the plate. In suchembodiments, it is preferred that the electrodes of a particular column(or set of columns or portions thereof) to be energized together beoperably connected such that when one electrode pair is energized, allof them are energized with energy having the same energizing parameters,A “portion of” the wells (or electrodes) of a column or row refers totwo or more, but fewer than all, of the wells (or electrodes) of thecolumn or row.

In other embodiments, the electroporation plates of the inventioncomprise one or more large volume chambers for conducting large-scaleelectroporation. In such embodiments, each chamber comprises a pluralityof electrifiable electrode sets that each minimally comprise at leasttwo electrodes. Each of the electrode sets can be independentlyenergized. As will be appreciated, such plates can be designed such thatthe plate and chamber(s) are manufactured as a single part.Alternatively, the plate may be designed to comprise two or more piecesthat can be assembled as needed or desired. For example, the plate maycomprise a support member manufactured to receive one or more largevolume chambers. In such embodiments, the chambers may be of the same ordifferent size, i.e., each chamber may have the capacity to hold thesame or a different volume of cells. In multi-piece assemblies, thesupport member may or may not contain electrical components. Inembodiments where the support member does not have electricalcomponents, the chamber units comprise the necessary circuitry andconnectors to allow the electrodes to be energized after connection to asuitable power supply (i.e., an electroporation pulse generator). Inembodiments where the support member does comprise certain of thecomponents necessary to energize the electrodes, the support member andchamber(s) comprise such connectors or other components as are necessaryto make the desired connections between the electrodes and a suitablepower supply. Of course, the invention contemplates a plethora ofpotential configurations for the plates of the invention. As such, theexact design of a plate according to the invention is left to thediscretion of the skilled artisan.

b. Solid Substrates.

Electroporation plates according to the invention can be made from anysuitable solid substrate. Preferred substrates are those that can bemanufactured to the desired specifications by molding processes and/ormachining. Particularly preferred are plastics or other polymers thatcan be formed into the desired shape by injection molding or similarprocesses. Polycarbonate, acrylonitrile butadiene styrene (ABS), andpolystyrene are particularly preferred this reason, although anymaterial (or combinations of different materials) that can be fashionedinto a large-scale electroporation according to the invention may beemployed. In embodiments that employ plastics, it will be appreciatedthat the plastics may be impregnated and/or reinforced with materials toprovide desired characteristics, e.g., improved rigidity, heatdissipation, insulation against electric current flow, etc.

Another advantage of plastics is the ability to use different colors anddiffering degrees of transparency in different parts of anelectroporation plate according to the invention. For example, manydetection systems are based on detecting light of specific wavelengths.For this reason, in many embodiments, the bottom of an electroporationplate is made from a transparent plastic, while portions of the platethat form the sides of the wells are made from an opaque plastic.

While plastics represent preferred examples of solid substrates forforming an electroporation plate according to the invention, otherembodiments employ ceramics or metals. As these materials, andtechniques for their preparation, are well known in the art, suchmaterials may be readily adapted for use in practicing the invention bythose ordinarily skilled in the art in light of this specification.

c. Electrodes.

Electrodes can be made or formed from any suitable electricallyconductive material, or combination of materials, including compositesof such materials. Preferably, the material(s) used for electrodes willbe biocompatible. When an electrode is formed from multiple materials(e.g., an electrically conductive material such as gold plated over anelectrically conductive material such as nickel plated over anelectrically conductive material such as copper, or a carrier doped witha electrically conductive material), it is preferred that at least theoutermost layer, i.e., the layer that will be exposed to a cellsuspension, is biocompatible. Preferred biocompatible electrodematerials include gold and titanium. Of course, other conductors mayalso be used (e.g., aluminum, various stainless steels, etc.), and theirselection is left to the discretion of the skilled artisan based on theparticular application.

Electrodes and conductors can be included in an electroporation plate byany suitable process. For example, in some embodiments, electrodematerials are plated or otherwise deposited onto the solid substrate indesired locations. In other embodiments, the solid substrate is machinedto accept electrodes for one or more pre-formed wells.

d. Manufacture.

A particularly preferred embodiment of a high throughput electroporationplate according to the invention (see FIG. 1) has been produced by amulti-step process, as follows. A polycarbonate frame providing thebasic structure for a 96-well electroporation plate was formed byinjection molding. The 96 wells were arranged in eight rows and twelvecolumns. The side walls of each well disposed in the direction of itsrow, as well as the top of the row adjacent to each well, was thenmolded with a material such as ABS or polycarbonate/ABS (PC-ABS) plasticblend to provide a plate having surface properties on certain of itssurfaces (here, the well side walls on the row sides of the well andtops of the rows) suitable for plating metals or other materials usefulas electrodes and conductors. After the wells were molded, the plate wasexposed to an etching solution to prepare the surfaces for application(e.g., by plating) of the electrode composition.

In this electroporation plate, three different layers of metal weresequentially deposited as relatively thin films on certain portions thesolid substrate (e.g., wells formed from ABS over a polycarbonate frame)using plating techniques that would differentiate between thepolycarbonate and ABS materials. Initially, a relatively thin copperfilm of a nominal thickness of about 10 microinches (mi) was plated ontothe ABS portions of the plate using electroless copper platingtechniques. Copper was chosen in this case because it adheres well toABS and provides transition bonding between the plastic and metallayers. Next, a relatively thick film of copper having a nominalthickness of about 1/1000 inch (1 mil) was deposited on the initialcopper layer using electroplating techniques. In plates such as these,these copper layers provide the bulk of current carrying capacity. Afterthe copper was applied, a thin film of nickel having a nominal thicknessof about 100 ml was deposited on the copper. Nickel was used because ofits conductive properties and because it adheres well to the copperlayer and forms a good substrate for electroplating gold. Finally, afinish layer of gold was plated on the nickel. Because of its relativecost, the gold was electrically plated using a controlled electroplatingtechnique. The gold layer had a nominal thickness of about 10 mi. Aswill be appreciated, due to cost considerations, the nominal thicknessof the copper and nickel layers can vary as much as 50% or more. Thethickness of the gold layer was more tightly controlled, and preferablyvaries less than about 5%. Electrodes useful in practicing theinvention, including multi-layer electrodes such as those describedabove, preferably have the capacity for use under a variety of differentelectroporation conditions (including 400 volts (V) for 10 milliseconds(msec) using a standard buffered saline solution), making a particularplate useful for a variety of different electroporation conditions.Indeed, given that some or all of the wells can be independentlyelectrically addressed (i.e., energized) as compared to other wells onthe plate, a single plate can be used to test a plurality of differentelectroporation conditions. For purposes of quality control, theelectrodes

Such three-layer electrodes as described above were calculated to havenominal track resistance of 0.18 ohm for the copper layer, 0.73 ohm forthe nickel layer, and 2.5 ohm for the gold layer, yielding a totalresistance of about 0.136 ohm. It is believed that about 75% of anapplied electric current (i.e., an electroporation pulse) will flowthrough the copper, although it will migrate into the nickel and thenthe gold layer to reach the sample.

After adding the electrode layers, a transparent, insulating bottom madeof plastic (e.g., polyester or polystyrene) was bonded to the otherportions of plate using an adhesive cured under ultraviolet light so asto ensure that the bottom of each well was completely sealed to preventwell-to-well leakage. The plate bottom may be clear, translucent, oropaque, as may other parts of the plates. It has been found that whenthe plates are to be used in luminescence-based assays, white bottomsare preferred. In any event, after a suitable bottom has been affixed tothe plate, the plate preferably is then sealed and sterilized.

In another particularly preferred embodiment of a high throughputelectroporation plate according to the invention, an insulating polymeris over-molded onto an array of electrically conductive electrodesconfigured so as to ultimately provide a plate having 96 wells arrangedin an 8×12 array such that each well contains at least a pair ofelectrodes, and wherein the electrodes of some or all of the wells maybe energized independently of the electrodes of other wells in theplate.

3. Applications.

Because the high throughput electroporation plates of the inventionprovide at least two wells that contain electroporation electrodes thatcan be independently energized or addressed, multiple electroporationexperiments can be performed in a single plate. Similarly, becausecertain large-scale electroporation plates according to the inventioncomprise one or more large volume electroporation chambers, each ofwhich contain two or more electrode sets that can be independentlyenergized or addressed, large volumes of cells can be efficientlyelectroporated in a single vessel. Moreover, existing electroporationpower supplies can be used more efficiently when used in conjunctionwith such plates, as it is now possible to energize different regions ofa plate, or different electrode sets of a single chamber, at differenttimes, thereby allowing the power supply to recharge betweenenergizings. Thus, electroporation conditions can be readily optimizedusing conventional pulse generators.

Another application of the invention involves introducing exogenouschemicals such as nucleic acid molecules into host organisms (such aseukaryotic and prokaryotic host cells), as such techniques are centralto many types of experiments, analyses, and therapies. For example, whensearching for a gene of interest in a DNA library, the library mustfirst be introduced into a suitable host cell population. Since atypical DNA library (e.g., a library for the genome of organism suchman, mouse, corn, etc.) is very complex (i.e., contains thousands, tensof thousands, or more different genes), the number of independent clonesneeded to completely represent each gene in the library is large. Tocreate a library that completely represents an organism's genome, forexample, the efficiency at which DNA can be introduced into the hostcells may become limiting. By optimizing this process, the ability tocreate and screen DNA libraries is facilitated.

Similarly, many other experimental analyses are limited by the abilityto introduce DNA into a host organism. When cloning large segments ofDNA for whole genome analysis (i.e., using bacterial artificialchromosomes), when performing cloning using the polymerase chainreaction (PCR), or when carrying out random mutagenesis of a gene,followed by cloning all potential altered forms, success often dependson the size of the initial transformation pool. Again, developingconditions that improve the process of introducing nucleic acids into ahost organism increases the chance that the experiment will succeed.

In addition to experimental uses, electroporation can be used fortherapeutic purposes. Here, the plates of the invention can be used tointroduce exogenous molecules having a therapeutic benefit into cells ofa patient, particularly in ex vivo formats. Examples of therapeuticexogenous molecules include nucleic acid molecules. Nucleic acids can bedelivered to effect so-called “gene therapy”, i.e., the introductioninto a patient of one or more genes intended to produce a therapeuticbenefit. Alternatively, the large-scale electroporation plates of theinvention can be used in an ex vivo format to introduce drugs from otherdrug classes (e.g., small molecule pharmaceuticals, therapeuticproteins, etc.) into cells of. After treatment, the cells may then beintroduced into a patient. Often, the cells will be reintroduced intothe patient from whom they were removed.

In developing and refining electroporation methodology, factors havebeen identified that impact the efficiency of the transfer of exogenousmolecules, e.g., nucleic acids. These factors include electrical fieldstrength, pulse decay time, pulse shape, reaction temperature, celltype, suspension buffer composition, and the concentration and size ofthe species (including more than one species) of exogenous molecule,e.g., a nucleic acid molecule, to be transferred. Given the number ofparameters that can influence the efficiency of an electroporationexperiment, in research and commercial settings it is often important todefine conditions that will result in high efficiency transfer of thedesired molecules (e.g., recombinant nucleic acids) into a particularhost cell line. Thus, optimization will be important to increasing theuse and reproducibility of electroporation in biomolecular studies.

To perform optimization, an electroporation system of the invention willtypically further comprise an optimization computer adapted to optimizeelectroporation conditions, alone or in conjunction with otherexperimental conditions, using electroporation data stored in thememory. Because a high throughput electroporation plate according to theinvention comprises a plurality of independently addressable wells, aplurality of electroporation experiments can be performed. Here, an“electroporation experiment” refers to the particular set of energizingparameters used to energize the electrodes in a given well (preferably,several wells). Thus, a plurality of different electroporationexperiments can be performed on a single plate. Analysis of theresulting data enables electroporation conditions (i.e., the energizingparameters and other conditions, e.g., buffer, cell concentration, etc.)to be optimized for a particular cell line or cell population.Optimization is preferably performed using statistical methods andtechniques such as multi-variate analysis so that optimalelectroporation conditions (or at least energizing parameters for thegiven host cell, buffer, and exogenous molecule) can be determined.

All patents and patent applications, publications, scientific articles,and other referenced materials mentioned in this specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each of which is hereby incorporated byreference to the same extent as if each reference had been incorporatedby reference in its entirety individually. Applicants reserve the rightto physically incorporate into this specification any and all materialsand information from any such patents and patent applications,publications, scientific articles, electronically available information,and other referenced materials or documents.

The specific electroporation plates, systems, and methods described inthis specification are representative of preferred embodiments and areexemplary and not intended as limitations on the scope of the invention.Other objects, aspects, and embodiments will occur to those skilled inthe art upon consideration of this specification and are encompassedwithin the spirit of the invention as defined by the scope of theclaims. It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, or limitation orlimitations, which is not specifically disclosed herein as essential.Also, the terms “comprising”, “including”, “containing”, etc. are to beread expansively and without limitation. It must be noted that as usedherein and in the appended claims, the singular forms “a”, “an”, and“the” include plural reference unless the context clearly dictatesotherwise.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any now-existing orlater-developed equivalent of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention as claimed. Thus, it will beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand/or variation of the disclosed elements may be resorted to by thoseskilled in the art, and that such modifications and variations arewithin the scope of the invention as claimed.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. An electroporation plate, comprising: a. a plurality of energizableelectroporation wells arrayed in a solid substrate, wherein eachelectroporation well comprises at least two electroporation electrodesdisposed therein, and wherein the at least two electroporationelectrodes in at least two of the wells can be independently energized;and b. electrical connections for connecting the electroporationelectrodes to an energy source.
 2. An electroporation plate according toclaim 1 that comprises about 2, 12, 24, 96, 192, 288, 384, 576, 768,672, 1536, 3072, or 6144 electroporation wells.
 3. An electroporationplate according to claim 1 wherein the electroporation wells are ofsubstantially uniform dimensions.
 4. An electroporation plate accordingto claim 1 wherein the electroporation wells are substantially eithercylindrical or rectangular.
 5. An electroporation plate according toclaim 1 wherein each electroporation well has a volume between about 1uL and about 10 mL.
 6. An electroporation plate according to claim 1wherein the electroporation wells have a volume of about 1 uL to about 1mL.
 7. An electroporation plate according to claim 1 wherein theelectroporation wells have a volume of about 1 mL to about 10 mL.
 8. Anelectroporation plate according to claim 1 wherein one of theelectroporation electrodes in an electroporation well is disposedopposite of the other electroporation electrode.
 9. An electroporationplate according to claim 1 wherein each electroporation well comprisesat least one sidewall and a bottom wall.
 10. An electroporation plateaccording to claim 9 wherein the electroporation electrodes of anelectroporation well are integrated into the sidewall.
 11. Anelectroporation plate according to claim 1 wherein the electroporationwells arranged in a plurality of rows and columns.
 12. Anelectroporation plate according to claim 11 wherein the electroporationelectrodes in the electroporation wells of a row are operably connectedso as to be simultaneously energized.
 13. An electroporation plateaccording to claim 12 wherein the operably connected electroporationelectrodes in the electroporation wells of one row can be energizedindependently of the electroporation electrodes in electroporation wellsin other rows of the electroporation plate.
 14. An electroporation plateaccording to claim 13 wherein the electroporation electrodes in each roware energized independently of the electroporation electrodes in otherrows of the electroporation plate.
 15. An electroporation plateaccording to claim 1 wherein the electrodes in each well comprise aplurality of electrodes that are each individually energizable.
 16. Anelectroporation plate according to claim 15 wherein said plurality ofelectrodes comprises between 2 and 12 pairs of electrodes.
 17. Anelectroporation plate according to claim 16 wherein the electrodes ofeach pair of electrodes comprise a cathode and an anode positioned insaid well opposite to one another.
 18. An electroporation plateaccording to claim 17 wherein at least two adjacent pairs of electrodesare energized simultaneously as an electrode set.
 19. An electroporationplate according to claim 1 wherein the electroporation electrodes ineach electroporation well are independently energizable.
 20. Anelectroporation plate according to claim 1 wherein the materialcomprising the solid substrate is selected from the group consisting ofplastic, metal, and ceramic.
 21. An electroporation plate according toclaim 1 wherein the solid substrate is transparent.
 22. Anelectroporation plate according to claim 1 wherein the solid substrateis translucent.
 23. An electroporation plate according to claim 1wherein the solid substrate is opaque.
 24. An electroporation plateaccording to claim 1 wherein the electroporation electrodes areintegrated in the solid substrate.
 25. An electroporation plateaccording to claim 1 wherein the electroporation electrodes aredeposited on a surface of the solid substrate.
 26. An electroporationplate according to claim 1 wherein the electroporation electrodes aredeposited by vapor deposition.
 27. An electroporation plate according toclaim 26 wherein the electroporation electrodes comprise an electricallyconductive material or a combination of electrically conductivematerials.
 28. An electroporation plate according to claim 26 whereinthe electrical connection comprises pins and sockets.
 29. Anelectroporation plate according to claim 26 wherein the electricalconnection comprises an independent electrical contact for each pair ofelectroporation electrodes that is independently energized.
 30. Anelectroporation system, comprising: a. an electroporation plateaccording to claim 1; and b. a power supply adapted for connection tothe electrical connector of the electroporation plate.
 31. Anelectroporation system according to claim 30 further comprising a platehandler configured to hold the electroporation plate during operation ofthe electroporation system.
 32. An electroporation system according toclaim 30 further comprising a plate reader.
 33. An electroporationsystem according to claim 32 wherein the plate reader uses a readingelement selected from the group consisting of a machine vision device, aspectrophotometer, and a luminomitor to collect electroporation data byexamining the contents of one or more of the electroporation wells ofthe electroporation plate.
 34. An electroporation system according toclaim 32 wherein the plate reader is integrated into the plate handler.35. An electroporation system according to claim 30 wherein the platehandler is a robotic plate handler.
 36. An electroporation systemaccording to claim 35 further comprising a plate storage facility forstoring electroporation plates during the course or following completionof an electroporation experiment.
 37. An electroporation systemaccording to claim 36 wherein the plate storage facility is an incubatorconfigured to hold a plurality of electroporation plates.
 38. Anelectroporation system according to claim 33 further comprising a datastorage device to store data collected by the plate reader.
 39. Anelectroporation system according to claim 38 further comprising anoptimization computer adapted to optimize electroporation conditionsfrom electroporation data stored in the memory.
 40. A method ofintroducing an exogenous molecule into a host cell, comprising usingelectroporation to introduce the exogenous molecule into the host cellin a suspension contained in an electroporation well of anelectroporation plate according to claim
 1. 41. A method according toclaim 40 wherein the exogenous molecule is a nucleic acid.
 42. A methodaccording to claim 40 wherein the host cell is selected from the groupconsisting of eukaryotic cell and a prokaryotic cell.
 43. A methodaccording to claim 42 wherein the host cell is a eukaryotic cellselected from the group consisting of an animal cell and a plant cell.44. A method according to claim 43 wherein the eukaryotic cell is ananimal cell selected from the group consisting of an mammalian cell, aninsect cell, a fish cell, a bird cell, an arachnid cell, a mollusk cell,and a crustacean cell.
 45. A method according to claim 44 wherein theeukaryotic cell is a mammalian cell from a mammal selected from thegroup consisting of bovine, canine, equine, feline, murine, ovine, andporcine animals.
 46. A method according to claim 43 wherein theeukaryotic cell is a cell from a mammalian cell line.
 47. A methodaccording to claim 43 wherein the eukaryotic cell is a plant cell from amonocotyledonous plant or a dicotyledonous plant.